ASH2L is the human homologue of Drosophila ASH2 (absent small homeotic 2) and represents a core member of the COMPASS and COMPASS-like complexes. Through interactions with WDR5 (WD-repeat protein 5) and RBBP5 (retinoblastoma binding protein 5), ASH2L activates SET1 domain-containing proteins (SET1A, SET1B and mixed lineage leukemia
(MLL)1-4) which subsequently catalyze H3K4 trimethylation . The presence of ASH2L is essential for optimal H3K4 trimethylation as knockdown of ASH2L led to a genome-wide decrease in H3K4me3 . Since COMPASS and COMPASS-like complexes are required for the transcriptional activation of numerous differentiation genes such as the HOX family, defects in ASH2L activity result in developmental defects [42, 63]. In mice, homozygous knockdown of ASH2L with gene-trap technology resulted in early embryonic lethality . ASH2L also promotes differentiation in muscle during later developmental stages. Through an interaction with ASH2L, PAX7 (paired box 7) recruits the WDR5-ASH2L-MLL2 complex to myogenic gene promoters and promotes trimethylation of H3K4 at these sites . MEF2D (myocyte enhancer factor 2D) is a transcription factor downstream of the p38 MAPK (mitogen-activated protein kinase) that also directs ASH2L-containing complexes to MyoD (myoblast determination protein)-bound genes in myoblasts . At specific loci, MyoD, PAX7, and ASH2L cooperate to induce a transcriptional program that leads to myogenic differentiation .
In addition to its role in development, ASH2L is also involved in tumor initiation. While ASH2L mRNA levels remain normal in human cancers, ASH2L protein levels increase dramatically in malignant cells, suggesting an oncogenic function for ASH2L . Supporting this hypothesis, ASH2L was also identified in complexes containing MYC (myelocytomato-sis viral oncogene homolog) oncogene . Since MYC activity increases in many types of cancers, the interaction between ASH2L and MYC suggests that ASH2L potentially adopts an oncogenic function . Indeed, ASH2L transforms primary rat embryo fibroblasts (REFs) through cooperation with H-Ras (Harvey rat sarcoma viral oncogene homolog) . As expected from an oncogene, knockdown of ASH2L reduces cell proliferation and inhibits transformation of REFs by MYC and H-RAS . A recent study revealed that ASH2L might affect PCa progression by acting as a co-activator of the androgen receptor (AR) . Co-immunoprecipitation experiments showed that AR interacts with ASH2L . Importantly, TrxG genes MLL1 and MLL2 also interact with AR , suggesting that ASH2L function in PCa results from association with complexes having H3K4 methyltransferase activity (See Figure 2A). Furthermore, siRNA (small interfering RNA) silencing of MLL or ASH2L significantly repressed AR signalling . However, pathways underlying the oncogenic nature of ASH2L remain poorly characterized. An important question that needs to be addressed is whether ASH2L promotes tumorigenesis through the same pathways in all tumor types or if its activity depends on the availability of other context-specific coregulators.
MENIN (protein encoded by multiple endocrine neoplasia 1 gene - MEN1) represents an integral subunit of the COMPASS-like complex that contains MLL1-2, MOF (MYST family his-tone acetyltransferases), and core COMPASS proteins that trimethylate H3K4 . In contrast to ASH2L, whether MENIN acts as an oncogene or a tumor suppressor highly depends on the specific tissue. Inherited mutations inactivating the MEN1 gene lead to a condition called multiple endocrine neoplasia type 1, in which the patients develop neoplasias in endocrine organs such as the parathyroid gland, the pituitary gland, and the pancreas [71,
72]. In endocrine organs, MENIN functions as a tumor suppressor and its role has been well characterized . MENIN induces the transcription of cyclin-dependent kinase inhibitors p18 and p27 . A mutated MEN1 gene therefore leads to a decrease in p18 and p27 expression, which accelerates cell-cycle progression. Loss of MENIN also promotes tumorigen-esis by releasing the inhibition of the oncogenic transcription factor JUN D (jun sarcoma virus 17 oncogene homolog) , which subsequently induces the expression of genes responsible for proliferation . In summary, mutation of the MEN1 gene leads to neoplasm formation in endocrine organs, which signifies that MENIN acts as a tumor suppressor in these tissues. However, studies in hematopoietic malignancies containing MLL fusion proteins suggest an oncogenic role for MENIN . In this context, MENIN binds to the MLL fusion protein and the complex activates the expression of key oncogenes which drive leu-kemogenesis . Since MLL fusion proteins do not possess a SET domain, it is important to note that the oncogenic function of MENIN does not implicate H3K4 methylation . Mis-regulation of MENIN activity also induces the formation of some solid tumors, although its mechanism of action varies considerably with the tumor type. For example, MENIN has been described as a tumor suppressor in non-small cell lung carcinomas (NSCLC) . MENIN function can also be observed in other solid tumors. In breast cancer, MENIN represents a transcriptional coactivator of ERa (estrogen receptor alpha). . In MCF7 breast cancer cells, MENIN co-localizes with ERa and activates ERa transactivation in a ligand-de-pendent manner . Interestingly, MLL2 was also independently shown to associate with ERa, suggesting that MENIN's oncogenic function requires the methyltransferase activity of its associated TrxG proteins . Furthermore, ER-positive breast cancer samples highly expressing MENIN had a worse outcome than those with low levels of MENIN after tamoxi-fen treatment . These findings support the idea that MENIN overexpression promotes the progression to a malignant phenotype in mammary tumors. As in breast cancer, MENIN seems to function as an oncoprotein in PCa . Significant upregulation of MENIN has been described in metastatic prostate tumors in comparison with their non-metastatic counterparts . Copy number gains for MEN1 represent frequent events in PCa and correlate with an increase in MENIN levels . Depletion of MENIN also significantly suppresses proliferation of DU145 PCa cells, in addition to increasing the levels of Integrin-p1, CAS-PASE8, and p53 tumor suppressor . Interestingly, MLL and MLL2 interact with AR. Since MENIN associates with MLL and MLL2, it is possible that its oncogenic function stems from cooperation with AR . Given these findings, we propose that MENIN promotes tumorigenesis in PCa.
MLL is a H3K4 methyltransferase and its role has been well characterized in certain types of leukemia where it is frequently involved in translocations . Five MLL family members, MLL1-5 are encoded in the mammalian genome . MLL and MLL2 can associate with MENIN, MOF and core TrxG subunits to form a complex with H3K4 and H4K16 methyltransferase activity . MLL3 and MLL4, on the other hand, can only be constituents of TrxG complexes that contain UTX and therefore possess H3K27 demethylase activity . MLL5 does not directly associate with core TrxG members and there is still no evidence that it has H3K4 methyltransferase activity . The oncogenic role of MLL in leukemia arises through a translocation that removes its SET domain responsible for H3K4 methylation . However, the role of MLL in PCa tumors has not been fully studied yet. Recent reports indicate that MLL enhances androgen signalling by directly interacting with AR and trimethy-lating H3K4 at AR target genes . In accordance with an activating role of MLL on AR signalling, RNAi-mediated depletion of MLL significantly decreases Prostate-Specific Antigen (PSA) levels . MLL expression is induced by SOX4 (Sex-determining region Y-box 4), a transcription factor that also activates epidermal growth factor receptor (EGFR), Integrin av, Ras-related C3 botulinum toxin substrate 1 (Rac1), and ADAM metallopeptidase domain 10 (ADAM10) . The pathways influenced by MLL activity suggest that MLL plays a role in promoting tumorigenesis. As is the case with MLL, MLL2 has also been shown to interact with AR. Although the role of MLL2 remains unclear in PCa, it seems to function as an oncoprotein in breast cancer . By acting as a coactivator, MLL2 stimulates the transcription of estrogen receptor (ER) target genes in ER+ breast tumors . Amplification of MLL2 has also been recorded in many solid malignancies including breast, pancreatic, brain, and ovarian tumors . In summary, it seems that the H3K4 methyltransferase activity of MLL1 and MLL2 mediates an oncogenic function in solid tumors.
The acetyltransferase MOF (males absent on the first) associates with MENIN, MLL or MLL2, and the core COMPASS proteins (ASH2L, DPY30, HCF1, RBBP5, and WDR5) to form a distinct TrxG complex . MOF specifically acetylates H4K16, a HPTM linked to transcriptional activation . In cancer cells, loss of H4K16ac represents a common event and correlates with general hypomethylation of repetitive DNA sequences . This suggests that MOF activity is inhibited in cancer cells and that MOF therefore functions as an oncosuppressor. Many important growth-regulatory pathways are regulated by MOF, some of which do not require the H3K4 methyltransferase ability of COMPASS-like complexes. First of all, MOF inhibits cancer progression by cooperating with forkhead box protein P3 (FOXP3) . FOXP3 recruits MOF and the H3K4 methyltrasferase complex close to the transcription start site of tumor suppressors . The synergistic effect of H3K4 trimethyla-tion by MLL1-2 and of H4K16 acetylation by MOF results in transcriptional activation of target loci. In addition to its regulatory function in transcription, MOF also plays an important role in the DNA damage response (DDR), more specifically in the repair of double-stranded breaks (DSBs) . In response to ATM (ataxia telangiectasia mutated) pathway activation, MOF gets recruited to chromatin where it acetylates H4K16 near DSBs . At sites of DSBs, MOF stimulates the activity of DNA-dependent protein kinases (DNA-PKcs), a critical component of non-homologous end-joining (NHEJ) . Interestingly, studies demonstrated that MOF inhibition also affects homologous recombination (HR) in addition to NHEJ . In short, depletion of MOF leads to a reduction in H4K16 acetylation and is associated with defective DNA repair and chromosomal aberrations following ionizing radiation . MOF also plays another critical role in DDR and apoptosis induction by acetylating the DNA-binding domain of p53 at lysine 120 . This modification leads to increased p53 stability and triggers p53-mediated apoptosis through the upregulation of pro-apoptotic genes . In summary, MOF acts as an important tumor suppressor in PCa through three distinct mechanisms: 1) cooperating with FOXP3 to induce the expression of oncosuppressors 2) recruiting DDR proteins at DSBs by acetylating H4K16 and 3) acetylating p53 on lys120, leading to the expression of pro-apoptotic genes (See Figure 3A).
UTX, also called KDM6 (histone lysine demethylase 6), associates with complexes containing the H3K4 methyltransferases MLL3 or MLL4 . UTX possesses H3K27 demethylase activity and therefore plays a prominent role in the balance between PcG-mediated repression and TrxG-mediated activation . The role of UTX has been well characterized in HOX gene regulation during embryonic development . When a cell receives a differentiation signal, UTX promotes HOX gene expression in two ways: 1) It interacts with MLL3 or MLL4, which catalyze the trimethylation of H3K4 at HOX loci and 2) It demethylates H3K27me3, a chemical modification associated with transcriptional repression . Aside from its role in development, UTX has also been linked to cancer where it functions as a tumor suppressor . The demethylase activity of UTX seems particularly relevant to PCa as PRC2 gain of function and H3K27 trimethylation represent common hallmarks of aggres sive solid tumors .This global increase in H3K27me3 implies a loss of function for UTX in PCa progression. UTX also counteracts PcG-mediated silencing by stimulating the ubiqui-tination of H2A, a HPTM associated with transcriptional activation . Moreover, UTX further antagonizes PcG function by interacting with BRM (ATP-dependent helicase brahma) and subsequently recruiting CBP (CREB-binding protein), which catalyzes H3K27 ace-tylation. The added acetyl group restricts the access to PRC2 at the modified sites and therefore inhibits PcG-induced silencing . UTX also plays an important role in repressing cellular proliferation through the regulation of RB levels . It promotes cell cycle arrest by upregulating RB, a commonly altered tumor suppressor that inhibits the transcription of genes responsible for G1/S transition . In summary, UTX represses many molecular processes associated with PCa initiation and progression (See Figure 3B). The tumor sup-pressive role of UTX has been validated in other tumor types. Systematic sequencing of renal carcinomas, multiple myelomas, medulloblastoma, and different types of leukemias all revealed inactivating mutations in a significant number of patients [107-111] Furthermore, UTX downregulation correlates with poor clinical outcome in breast cancer . Given the prominence of PcG in PCa, inactivation of UTX most likely represents a critical event in the progression to metastasis.
WDR5 represents a core member of the COMPASS and COMPASS-like complexes whose functional role in cancer remains unclear . To date, very few studies have focused solely on the link between WDR5 and oncogenesis. However, WDR5 appears to have a promi nent role in embryogenesis. In ESCs, WDR5 interacts with the transcription factors OCT4 (octamer-binding transcription factor-4), SOX2, and NANOG to induce the expression of genes necessary for pluripotency and self-renewal . This transactivational ability correlates with H3K4 trimethylation at the target loci. Furthermore, somatic cell reprogramming and formation of induced pluripotent stem cells (iPSCs) also requires the presence of WDR5 . WDR5 has been shown to be essential for proper HOX gene activation as Xenopus Lae-vis tadpoles exhibit a wide range of developmental defects upon WDR5 depletion . Moreover, WDR5 cooperates with the canonical Wnt pathway to induce osteoblast and chondrocyte differentiation . WDR5 is expressed upon bone morphogenetic protein (BMP) signalling, another pathway associated with differentiation . In fact, WDR5 was initially called ''BMP-2-induced gene 3 kb'' and subsequently changed to its current name .
Recently, a study demonstrated that WDR5 is induced under hypoxic conditions and is required for epithelial-mesenchymal transition (EMT) . Hypoxia activates the expression of WDR5 and HDAC3. WDR5 and H3K4 methyltransferase complexes are then recruited to promoters of mesenchymal genes to activate their transcription . In parallel, HDAC3 removes pre-existing acetyl groups from H3K4 to potentiate WDR5 action. HDAC3 also removes histone acetylation marks from promoters of epithelial genes, further pushing the cell towards a mesenchymal phenotype . EMT represents an essential step for tumor metastasis [120-122]. Since WDR5 is required for EMT, WDR5 could potentially act as an onco-gene by promoting metastasis of primary prostate tumors (Figure 2B). Although the oncogenic role of WDR5 has not been tested in PCa, studies in head and neck squamous cell carcinoma showed that coexpression of HIF-1a, WDR5, and HDAC3 is associated with metastasis and poor prognosis . These results suggest that WDR5 functions as an onco-protein by triggering EMT. However, further studies are needed to assess the consequences of WDR5 expression in PCa.
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